Cysteine scanning mutagenesis of the noncatalytic nucleotide binding site of the yeast V-ATPase

To investigate residues involved in the formation of the noncatalytic nucle otide binding sites of the vacuolar proton-translocating adenosine triphosp hatase (V-ATPase), cysteine scanning mutagenesis of the VMA2 gene that enco des the B subunit in yeast was performed. Replacement of the single endogen ous cysteine residue at position 188 gave rise to a Cys-less form of the B subunit (Vma2p) which had near wild-type levels of activity and which was u sed in the construction of 16 single cysteine-containing mutants. The abili ty of adenine nucleotides to prevent reaction of the introduced cysteine re sidues with the sulfhydryl reagent 3-(N-maleimidopropionyl)biocytin (biotin -maleimide) was evaluated by Western blot. Biotin-maleimide labeling of the purified V-ATPase from the wild-type and the mutants S152C, L178C, N181C, A184C, and T279C was reduced after reaction with the nucleotide analog 3'-O -(4-benzoyl)benzoyladenosine 5'-triphosphate (BzATP). These results suggest the proximity of these residues to the nucleotide binding site on the B su bunit, In addition, we have examined the level of endogenous nucleotide bou nd to the wild-type V-ATPase and to a mutant (the A subunit mutant R483Q) w hich is postulated to be altered at the noncatalytic site and which display s a marked nonlinearity in ATP hydrolysis (MacLeod, K. J., Vasilyeva, E., B aleja, J. D., and Forgac, M. (1998) J. Biol. Chem. 273, 150-156), The R483Q mutant contained 2.6 mol of ATP/mol of V-ATPase compared with the wild-typ e enzyme, which contained 0.8 mol of ATP/mol of V-ATPase, These results sug gest that binding of additional ATP to the noncatalytic sites may modulate the catalytic activity of the enzyme.